Late assembly of the Vibrio cholerae cell division machinery postpones

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1 2 Late assembly of the Vibrio cholerae cell division machinery postpones septation to the last % of the cell cycle 3 4 Elisa Galli, Evelyne Paly and François-Xavier Barre,# Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CRS, Université Paris Sud, France

2 Supplementary Figure. a. Cells were grown overnight in LB at 3 C. After normalization for cell density, each culture was serially diluted. 5 μl of each dilution was spotted on a LB plate incubated overnight either at 3 C or at 42 C and photographed. b. Phase-contrast images of V. cholerae 696 cells carrying the ftsz ts or ftsi ts allele after h and 3min growth at 42 C. c. Combined phasecontrast and fluorescent image of ftsi ts V. cholerae cells ectopically expressing YGFP-FtsI at 42 C. Scale bar = 2 μm. Supplementary Figure 2. a. Cellular localisation of FtsZ-RFPT, YGFP-FtsA, RFPT-ZapA, FtsK-YGFP, YGFP-FtsL, sfgfp-ftsi, sfgfp-fts, DsbAss-mCherry- SPR and HubP-sfGFP in V. cholerae 696 cells. Scale bar = 2 μm. b. Phasecontrast image and cellular localisation of YGFP-FtsA, RFPT-ZapA, FtsK-YGFP, YGFP-FtsL, sfgfp-ftsi and sfgfp-fts in cephalexin treated cells. Scale bar = 2 μm. c. Percentage of minicells formed at each division in LB as determined from 2 independent time-lapse experiments. hubp-sfgfp is integrated at hubp native locus and expressed from the native promoter. Error bars represent standard deviation. d. Cellular localisation of HubP-YFP ectopically expressed from a PBAD promoter in addition to hubp wild-type copy. Scale bar = 2 μm. e. Demograph of V. cholerae 696 cells ectopically expressing HubP-YFP from a PBAD promoter in addition to hubp wild-type copy. Cells were not oriented. Supplementary Figure 3. Theoretical deduction of the cell cycle advancement of cells using their length. a. V. cholerae cell length increases exponentially with cell cycle. Log scale representation of cell length as a function of cell cycle advancement for individual V. cholerae cells grown on a % (w/v) agarose pad in M9 minimal medium,.2% fructose and μg/ml thiamine. Blue circles: results for 25 complete lineages, i.e. for cells in which both birth and division were 2

3 observed; red line: median log of the length as a function of the cell cycle; black line: linear regression; M: length at division; m: length at birth. b. Scheme depicting the relationship between cell cycle advancement and cell length. At 5% of the cell cycle, cell elongation is smaller than half of the total elongation from birth to division. M; division length; m: birth length; (M+m)/2: half of the total elongation from birth to division. c. Cell length distribution of 33 V. cholerae cells ectopically producing a fluorescently tagged copy of FtsZ. M: largest observed cell length; m: smallest observed cell length. Marks showing the theoretical cell cycle stages are shown as black (.-.4,.6-.9) and red (,.,.5,.,) ticks. Tick positions were determined using the exponential relationship between cell length and cell cycle and assuming that M and m correspond to the division length and the birth length. d. FtsZ-localisation in demograph representation. Marks showing the theoretical cell cycle stages are shown as black (.2-.4,.6,.8) and red (,.,.5,.,) ticks. Each cell cycle stage tick is located at the projection profile of the longest cell whose length is below the theoretical length reached at this particular stage. e. Cell length distribution of 784 V. cholerae cells ectopically producing a fluorescently tagged copy of FtsK. M: largest observed cell length; m: smallest observed cell length. Marks showing the theoretical cell cycle stages are shown as black (.-.4,.6-.9) and red (,.,.5,.,) ticks. Tick positions were determined using the exponential relationship between cell length and cell cycle and assuming that M and m correspond to the division length and the birth length. f. FtsK-localisation in demograph representation. Marks showing the theoretical cell cycle stages are shown as black (.2-.4,.6,.8) and red (,.,.5,.,) ticks. Each cell cycle stage tick is located at the projection profile of the longest cell whose length is 3

4 below the theoretical length reached at this particular stage. Supplementary Figure 4. Examples of time-lapse individual lineages in the and representation., old pole;, new pole. a. FtsZ-RFPT. b. YGFP- FtsA. c. RFPT-ZapA. d. FtsK-YGFP. e. DsbAss-mCherry-SPR. Supplementary Figure 5. a. cell cycle distribution of DsbAss-mCherry- SPR (compilation of 44 single cell cycles). b. Examples (i, ii) of time-lapse images of 696 cells expressing YGFP-FtsK and DsbAss-mCherry-SPR. ne frame was taken every 3 minutes. n the top-right corner of each frame is indicated the time in minutes from the beginning of imaging. White arrows show the arrival of tagged proteins to mid-cell. Scale bar = 2 μm. Supplementary Figure 6. a. cell cycle distribution of HubP-sfGFP (compilation of 88 single cell cycles), YGFP-ParB (compilation of 69 single cell cycles) and oric locus (compilation of 5 single cell cycles). b. cell cycle distribution of YGFP-FtsK (compilation of 73 single cell cycles) in V. cholerae 696 ΔhubP cells. c. Time-lapse images of 696 cells expressing DsbAssmCherry-SPR and HubP-YFP, ectopically expressed from a PBAD promoter in addition to hubp wild-type copy. ne frame was taken every 4 minutes. n the top-right corner of each frame is indicated the time in minutes from the beginning of imaging. White arrows mark proteins arrival at the division sites. Scale bar = 2 μm. Supplementary Figure 7. Examples of time-lapse individual lineages in the and representation., old pole;, new pole. a. HubP-sfGFP. b. YGFP- ParB. c. Movement of oric followed by inserting a lac array in its proximity and expressing a LacI-YGFP fusion. Supplementary Table. List of strains and plasmids used in this study. 4

5 Movie. Examples of FtsZ-RFPT time-lapses in V. cholerae cells. ne frame was taken every 3 minutes. Cells were grown in M9 minimal medium supplemented with.2% fructose and μg/ml thiamine at 3 C. Movies start from the beginning of imaging. Movie 2. Time-lapse of FtsZ-RFPT and FtsK-YGFP localisation in V. cholerae cells. ne frame was taken every 4 minutes. Cells were grown in M9 minimal medium supplemented with.2% fructose and μg/ml thiamine at 3 C. μg/ml cephalexin was added to the agarose slide. Movie 3. Time-lapse of FtsK-YGFP and DsbAss-mCherry-SPR localisation in V. cholerae cells. ne frame was taken every 3 minutes. Cells were grown in M9 minimal medium supplemented with.2% fructose and μg/ml thiamine at 3 C. μg/ml cephalexin was added to the agarose slide. Movie 4. Time-lapse of FtsK-YGFP and HubP-RFPT localisation in V. cholerae cells. ne frame was taken every 3 minutes. Cells were grown in M9 minimal medium supplemented with.2% fructose and μg/ml thiamine at 3 C. μg/ml cephalexin was added to the agarose slide. Movie 5. Time-lapse of DsbAss-mCherry-SPR and HubP-sfGFP localisation in V. cholerae cells. ne frame was taken every 3 minutes. Cells were grown in M9 minimal medium supplemented with.2% fructose and μg/ml thiamine at 3 C. μg/ml cephalexin was added to the agarose slide. Movie 6. Time-lapse of DsbAss-mCherry-SPR and HubP-YFP localisation in V. cholerae cells. HubP-YFP was ectopically expressed from a PBAD promoter in addition to hubp wild-type copy. ne frame was taken every 4 minutes. Cells were grown in M9 minimal medium supplemented with.2% fructose and μg/ml thiamine at 3 C. μg/ml cephalexin was added to the agarose slide. 5

6 a b dilutions ftsz ts 42 C wt 3 C ftsz ts ftsi ts ftsi ts 42 C wt ftsz ts ftsi ts c 42 C ftsi ts + YGFP-FtsI 42 C Supplementary Figure

7 a FtsZ FtsA ZapA FtsK FtsL FtsI Fts SPR HubP A b Cephalexin-treated V. cholerae cells FtsA FtsK Phase contrast ZapA FtsL d c e 5 5 minicell per divisions (%) Pbad HubP-YFP FtsI Cell length Supplementary Figure 2 Fts Pbad HubP-YFP

8 a M b M Cell Length (log) Cell Length m+m 2 m..5. Cell Cycle m..5. Cell Cycle c 8 FtsZ d..3 FtsZ number of cells Cell Cycle.4.5 m Cell Length M Cell Cycle..8 e f FtsK 2 FtsK..3 number of cells Cell Cycle.4.5 m Cell Length M..5. Cell Cycle.6..8 Supplementary Figure 3

9 a FtsZ b FtsA c ZapA d FtsK e SPR Cell cycle progression (%) Cell cycle progression (%) Cell cycle progression (%) Cell cycle progression (%) Cell cycle progression (%) Supplementary Figure 4

10 a SPR, IStime Cell length 5 Cell cycle SPR FtsK b i SPR FtsK ii Supplementary Figure

11 a HubP, IStime ParB, IStime Cell length oric, IStime Cell cycle..5.. Cell cycle..5 Cell cycle FtsK in hubp, IScycle b Cell length. c..5 Cell cycle Pbad:: HubP SPR. Supplementary Figure

12 a HubP b ParB c oric Cell cycle progression (%) Cell cycle progression (%) Cell cycle progression (%) Cell cycle progression (%) Cell cycle progression (%) Supplementary Figure 7

13 Supplementary Table. List of bacterial strains and plasmids Strains ame Relevant genotype or features Reference EGV 696 ChapR ΔlacZ::(P BAD ::ftsz-rfpt-sh ble) zeo R gm R () EGV 696 ChapR ΔlacZ::(P BAD ::RFPT-zapA-Sh ble) zeo R gm R This study EGV ΔlacZ::(P BAD ::ftsz-rfpt-sh ble) ΔhapR::(P lac ::laci- YGFP-cat) + lac array-aph inserted on chr at position.5 Mb (53355 bp) (oric) zeo R gm R cml R kan R This study EGV ChapR ΔlacZ P ftsk ::ftsk-ygfp-sh ble zeo R gm R () EGV ΔlacZ P ftsk ::ftsk-ygfp-sh ble ΔhapR::(P BAD ::ftsz-rfptcat) () R zeo R gm R cml EGV ChapR ΔlacZ mincd::sh ble zeo R gm R () EGV ΔlacZ::(P BAD ::YGFP-ftsA-Sh ble) zeo R gm R This study EGV ChapR ΔlacZ::(P lac ::YGFP-parB-Sh ble) zeo R gm R This study EGV ChapR ΔlacZ::(P BAD ::YGFP-ftsL-Sh ble) zeo R gm R This study EGV 696 ChapR ΔlacZ ΔhubP mincd::sh ble zeo R gm R () EGV ChapR ΔlacZ::(P BAD ::dsba ss -mcherry-spr-sh ble) This study SPR domain of E. coli Fts (Fts[243-39]) (signal sequence from DsbA) zeo R gm R EGV ChapR ΔlacZ::(P BAD ::hubp-yfp-sh ble) zeo R gm R This study EGV ChapR ΔlacZ::(P BAD ::RFPT-zapA-Sh ble) carrying the ftsz This study ts chromosomal mutation (G6S) zeo R gm R EGV ChapR ΔlacZ P ftsk ::ftsk-ygfp-sh ble carrying the ftsz ts This study chromosomal mutation (G6S) zeo R rif R gm R EGV ChapR ΔlacZ::(P BAD ::YGFP-ftsA-Sh ble) carrying the ftsz This study ts chromosomal mutation (G6S) zeo R gm R EGV ChapR ΔlacZ::(P BAD ::YGFP-ftsL-Sh ble) carrying the ftsz This study ts chromosomal mutation (G6S) zeo R gm R EGV ChapR ΔlacZ::(P BAD ::YGFP-ftsI-Sh ble) carrying the ftsz This study ts chromosomal mutation (G6S) zeo R gm R EGV ChapR ΔlacZ::(P BAD ::dsba ss -mcherry-spr-sh ble) carrying the ftsz ts chromosomal mutation (G6S) SPR domain of E. coli Fts (Fts[243-39]) (signal sequence from DsbA) zeo R gm R This study EGV ΔlacZ P ftsk ::ftsk-ygfp-sh ble ΔhapR::(P BAD ::dsba ss - mcherry-spr-cat) SPR domain of E. coli Fts (Fts[243-39]) (signal sequence from DsbA) zeo R gm R cml R EGV ΔlacZ::(P BAD ::hubp-yfp-sh ble) ΔhapR::(P BAD ::dsba ss - mcherry-spr-cat) SPR domain of E. coli Fts (Fts[243-39]) (signal sequence from DsbA) zeo R gm R cml R This study This study EGV ChapR ΔlacZ P ftsk ::ftsk-ygfp-sh ble P hubp ::hubp- This study RFPT-aadA zeo R spec R gm R EGV ChapR ΔlacZ P hubp ::hubp-rfpt-aada carrying the ftsz ts This study chromosomal mutation (G6S) spec R rif R gm R EGV ChapR ΔlacZ::(P lac ::sfgfp-fts-sh ble) carrying the ftsz This study ts chromosomal mutation (G6S) zeo R gm R EGV ChapR ΔlacZ ΔhubP P ftsk ::ftsk-ygfp-sh ble zeo R gm R This study EPV5 696 ChapR ΔlacZ gm R (2) EPV ChapR ΔlacZ carrying the ftsz ts chromosomal mutation () (G6S) rif R gm R EPV ChapR ΔlacZ::(P lac ::sfgfp-fts-sh ble) zeo R gm R This study EPV432 EPV437 EPV ChapR ΔlacZ carrying the ftsi ts chromosomal mutation (Y388D) rif R gm R 696 ChapR ΔlacZ::(P BAD ::ftsz-rfpt-sh ble) carrying the ftsi ts chromosomal mutation (Y388D) zeo R gm R 696 ChapR ΔlacZ::(P BAD ::dsba ss -mcherry-spr-sh ble) carrying the ftsi ts chromosomal mutation (Y388D) SPR domain This study This study This study

14 of E. coli Fts (Fts[243-39]) (signal sequence from DsbA) zeo R gm R EPV ChapR ΔlacZ P hubp ::hubp-rfpt-aada carrying the ftsi ts This study chromosomal mutation (Y388D) spec R rif R gm R EPV ChapR ΔlacZ::(P lac ::sfgfp-ftsi-sh ble) zeo R gm R This study EPV ChapR ΔlacZ::(P lac ::sfgfp-ftsi-sh ble) carrying the FtsI ts This study chromosomal mutation (Y388D) zeo R gm R EPV ChapR ΔlacZ P hubp ::hubp-sfgfp gm R This study EPV ChapR P hubp ::hubp-sfgfp ΔlacZ::(P BAD ::dsba ss -mcherry- This study SPR-Sh ble) SPR domain of E. coli Fts (Fts[243-39]) (signal sequence from DsbA) zeo R gm R EPV ChapR ΔlacZ P hubp ::hubp-sfgfp mincd::sh ble zeo R gm R This study Plasmids (2) () () ame Relevant genotype or features Reference pad2 integration-excision vector; Tet -lac array-aph - Tet; sacb; ori R6K; cml R, kan R peg233 P BAD ::ftsz-rfpt-sh ble flanked by the upstream and downstream peg234 P BAD ::RFPT-zapA-Sh ble flanked by the upstream and downstream peg242 P BAD ::YGFP-ftsI-Sh ble flanked by the upstream and downstream This study peg245 P lac ::laci-ygfp-cat flanked by the upstream and downstream () regions of hapr; ori puc; amp R, cml R peg248 ftsk-ygfp-sh ble flanked by the upstream and downstream regions () of ftsk; ori psc; amp R, zeo R peg2 Sh ble flanked by the upstream and downstream regions of mincd; () ori puc; amp R, zeo R peg266 Sh ble flanked by the upstream and downstream regions of hubp; This study ori puc; amp R, zeo R peg27 P BAD ::YGFP-ftsA-Sh ble flanked by the upstream and downstream This study peg282 P lac ::YGFP-parB-Sh ble flanked by the upstream and downstream This study peg287 P BAD ::YGFP-ftsL-Sh ble flanked by the upstream and downstream This study peg323 P BAD ::dsba ss -mcherry-spr-sh ble flanked by the upstream and This study downstream regions of lacz; SPR domain of E. coli Fts (Fts[243-39]) (signal sequence from DsbA); ori puc; amp R, zeo R peg324 P BAD ::hubp-yfp-sh ble flanked by the upstream and downstream This study peg392 P BAD ::dsba ss -mcherry-spr-cat flanked by the upstream and This study downstream regions of hapr; SPR domain of E. coli Fts (Fts[243-39]) (signal sequence from DsbA); ori puc; amp R, cml R peg396 P lac ::sfgfp-fts-sh ble flanked by the upstream and downstream This study peg398 P lac ::sfgfp-ftsi-sh ble flanked by the upstream and downstream This study peg4 integration-excision vector; hubp-rfpt-aada at native locus; This study sacb; ori R6K; amp R, spec R peyy24 integration-excision vector; hubp-sfgfp at native locus; sacb; ori R6K; amp R Y. Yamaichi gift

15 References. Galli E, Poidevin M, Le Bars R, Desfontaines J-M, Muresan L, Paly E, Yamaichi Y, Barre F-X. 26. Cell division licensing in the multichromosomal Vibrio cholerae bacterium. at Microbiol : David A, Demarre G, Muresan L, Paly E, Barre F-X, Possoz C. 24. The two Cis-acting sites, pars and oric, contribute to the longitudinal organisation of Vibrio cholerae chromosome I. PLoS Genet :e4448.